It is now well appreciated that the skin is under daily assault from various environmental and lifestyle factors. A short list of these factors includes UVA radiation, UVB radiation, pollution, cigarette smoke, poor diet, insufficient rest, and psychological stress. This assault manifests as damage to skin cell DNA and proteins. Lines and wrinkles in the skin are among the less serious outcomes of skin cell damage, while melanoma is one of the more serious.
DNA Damage
Some environmental and lifestyle factors interact directly with skin cell DNA and/or proteins to cause damage, some factors cause damage indirectly, and some factors may do both. An example of direct DNA damage would be when skin cell DNA absorbs photons from the UVB part of the spectrum. The absorption of photons may cause mutations in the DNA sequence. For example, about 8% of all melanoma occurrence is due to direct DNA mutation.
An example of indirect damage to skin cell DNA and proteins is seen when ultraviolet photons enter the skin and are absorbed by chromophores. In an excited state, chromophores enter into reactions that lead to the formation of reactive oxygen species. For example, in human skin, UVB exposure is associated with the production of hydrogen peroxide, while UVA is associated with production of singlet oxygen. If the skin is unable to maintain homeostasis by neutralizing the reactive species, then the reactive species will damage skin cell DNA and proteins, through oxidation. This damage to DNA and proteins is called oxidative stress and is a major cause of skin aging. Also, about 92% of all melanoma occurrence is due to indirect, oxidative DNA damage.
DNA Damage: Prevention and Containment Verses Repair
DNA containing skin cells include keratinocyte stem cells and melanocytes. It is estimated that a single sun burn results in hundreds of thousands of DNA mutagenic base modifications such as T-T (thiamine-thiamine) dimers; 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-DG); 06MeG (06-methyl guanine); cyclobutane pyrimidine dimers (CPD); and 6-4 photoproducts (6-4PP) in affected cells. These mutations trigger various responses within the skin cells, which may lead to prevention of further DNA damage or apoptosis of the cell. For example, melanin production in melanocytes is known to be upregulated by UV radiation, as a protective measure. To deal with the threat from UV, melanin is produced by melanocytes in the lower epidermis, and with the help of outwardly migrating keratinocytes, is distributed throughout the upper and lower epidermis. In the first instance melanin seems to prevent further UV-induced DNA damage by acting as a UV filter that limits the amount of UV that penetrates to the lower epidermis, where melanocytes and keratinocyte stem cells are located. However, as a second line of defense, melanin contains DNA damage due to UV radiation by inducing apoptosis of keratinocytes. By promoting apoptosis, damaged DNA is contained, having less chance of being passed on to daughter cells (see, for example, Yamaguchi et al., Melanin mediated apoptosis of epidermal cells damaged by ultraviolet radiation: factors influencing the incidence of skin cancer; Arch Dermatol Res. 2008 April; 300 Suppl 1: S43-S50).
Apart from preventing and containing DNA damage, healthy keratinocytes and melanocytes have a natural internal mechanism for repairing DNA lesions. These mechanisms are different than those of prevention and containment, which involve different (although sometimes overlapping) reaction cascades. Compositions and methods of the present invention are foremost concerned with repair of DNA damage.
Cellular repair mechanisms exist to repair DNA lesions from various causes, not just the UV-induced damage that we have been discussing. However, repair of DNA lesions takes time. For example, repair of TT dimers and 6-4PP damage formed by UVB exposure may take up to 48 and 8 hours respectively, if not accelerated by an exogenous influence. Repair of 8-oxo-dG and 06MeG lesions due to UVA or UVB exposure, ozone, or smoke and pollution may take up to 2 hours. Ideally, DNA lesions are repaired before cell division occurs. If not, the preferred outcome is apoptosis. But if the DNA damage happens to adversely affect the upregulation of apoptosis, or if the mutation goes undetected, then the damaged DNA may be passed on to the next generation. Compositions and methods of the present invention are foremost concerned with maximizing the repair of DNA damage before cell division occurs.
A Cell's Preventative and Repair Mechanisms are Controlled by a Circadian Clock
Under normal conditions, cellular functions, including DNA expression and repair, do not occur at random times with equal probability. Rather, each cell has an endogenous cycle (or clock) of about 24 hours (i.e. a circadian rhythm), and the activities of the cell are regulated by this endogenous cycle. Absent some external stimulus, each cell would free-run according to its endogenous clock. However, the endogenous clocks of cells throughout the body are synchronized. In order to synchronize the activities of cells with each other and with the environment, the body is able to accept cues from the environment. The most notable environmental cue is the presence of absence of daylight. Thus, the circadian cycle consists of light and dark phases that roughly coincide with the phases of the solar day.
It is now appreciated that circadian rhythms allow cells to anticipate changes in the environment that might affect the cells, and to adapt to those changes in a timely fashion. As long as the genetic machinery of cellular circadian rhythms are functioning properly, cells carry out each of their many functions in a synchronized manner, at a time that is optimal for cell viability and/or homeostasis. For example, as daylight approaches (but even before the skin is under assault from UV), certain genes are activated to produce proteins that protect the cells against anticipated UV radiation damage. Then, as daylight wanes, these genes are turned off. On the other hand, the circadian genes themselves may be subject to attack by environmental factors. Damage to one or more genes that regulate a cell's circadian rhythm can put a cell out of sync with the environment and with other cells.
The Core Circadian Mechanism
Transcription factors are proteins that bind to specific DNA sequences to control the transfer of genetic information from DNA to RNA. The core circadian mechanism, or “cell clock”, is comprised of transcription factors that participate in out-of-phase, negative and positive feedback loops, that lead to oscillating gene transcription.
In the main negative feedback loop of mammals, a heterodimer of CLOCK and BMAL1 transcription factors activates transcription of the period (per) and cryptochrome (cry) genes. In humans, the period gene is actually a family of three genes per1, per2, and per3 and the cry gene family includes of cry1 and cry2. Following their translation, the PER and CRY proteins migrate into the cytoplasm and form PER/CRY complexes. Posttranslational regulation creates an intentional delay after which the PER/CRY complexes relocate into the cell nucleus. The concentrations of PER and CRY in the nucleus peak at the end of circadian daytime, at which time the CRY proteins then act to inhibit transcriptional activity of the CLOCK/BMAL1 heterodimer. Thus, CRY seems to turn off its own transcription.
On the other hand, in one positive feedback loop, PER2 upon translocating to the nucleus, seems to upregulate the transcription of BMAL1, eventually leading to the transcription of period and cry genes. Also, the CLOCK/BMAL1 dimer seems to upregulate rev-erbα and rora genes. Rora activates BMAL1 transcription, while rev-erbα suppresses CLOCK and BMAL1. The peak activities of the so called “canonical clock genes” (clock, bmal1, per1, per2, per3, cry1 and cry2) are out of phase, such that a self-sustaining loop results, having a period of approximately 24 hours.
The Cell Cycle
The cell cycle refers to the series of events that takes place in a cell leading to division and replication of the cell. The cycle is usually described as four or five sequential phases requiring about 24 hours to complete. Within each phase of the cell cycle, there are checkpoints that ensure that all requisite processes of a given phase are completed prior to initiation of the next phase. In human cells, the “first” phase is the Synthesis (S) phase in which the DNA of a cell is copied and synthesized. The S phase may typically last from 6-8 hours. In the G2 phase, lasting 3-4 hours, proteins are synthesized and the cell doubles in size. During Mitosis (M), the nuclear envelope breaks down so that each copy of the genetic material can separate to opposite poles of the cell. Following the formation of a new nuclear envelope around each set of chromosomes, the cell is pinched in two (cytokinesis). The M phase is about 1 hour long. The fourth and longest phase (6-12 hours) is GI which is characterized by RNA and protein synthesis. From the G1 phase, a cell may again enter the S phase or it may enter the G0 phase. In G0, the cell is quiescent. G0 may last for days or years. Stem cells may return from the G0 phase, entering at G1. Differentiated cells do not generally return from G0. Also, cells with damaged DNA may enter G0, rather than apoptosis.
Cell Cycle Checkpoints Inhibit Damaged DNA from Being Passed On
During cell division, checkpoints are used to regulate the progress of the cell through the cell cycle. Checkpoints prevent a cell from progressing to the next phase until completion of all necessary processes, including any repair of damaged DNA. In this way, checkpoints ensure that damaged or incomplete DNA is not passed on to daughter cells. Several checkpoints exist. The G1/S checkpoint (the Restriction checkpoint) interrupts the cell cycle so that the “decision” can be made to enter the quiescent phase or not. At the G2/M checkpoint the cell cycle is halted if damaged DNA is detected, which is not unusual. The Postreplication checkpoint concerns damaged DNA that has been replicated in the Synthesis phase. The replication of damaged DNA triggers a cellular response that prevents cell cycle progression until postreplication repair processes are completed. In human cells, the postreplication checkpoint makes time for repair by delaying the onset of the Mitosis phase. The chk1 gene exerts control over the postreplication checkpoint, while the p53 gene plays an important role in triggering the control mechanisms at both G1/S and G2/M checkpoints.
The Circadian Clock Also Regulates Cell Proliferation
In recent years, the importance of circadian clock in regulating cell proliferation has been appreciated. For example:                “Disruption of circadian timing . . . has far reaching consequences for normal regulation of cell division.” (Reddy et al., 2005 Circadian clocks: neural and peripheral pacemakers that impact upon the cell division cycle. Mutation Research 574 76-91).        “Detailed insight into the mechanisms whereby clock components interact with the cell cycle regulatory machinery has come from recent mouse studies. For example, . . . the delay between removal of liver tissue (partial hepatectomy) and the subsequent first wave of mitosis depends upon the time of day that the surgery was performed.” (Vallone et al., 2007 Start the clock! Circadian rhythms and development. Developmental Dynamics 236 142-155).        “From cyanobacteria to higher vertebrates, there are many examples of the circadian clock “gating” S-phase and mitosis of the cell cycle to occur during the night period.” (Vallone et al.). [Presumably, DNA synthesis and mitosis occur at night to protect DNA from harmful UV or other ionizing radiation from the sun.]        
Thus, the circadian clock exerts an overarching influence on the cellular cycle of DNA and protein synthesis, mitosis and cytokinesis, RNA synthesis and repair. Therefore, when environmental factors interfere with a cell's circadian mechanism, cellular function is compromised. We postulate that when treatment can entrain or resynchronize a cell's circadian clock, or when treatment can restore “normal” levels of circadian gene expression, then cellular functioning may be improved, cell damage may be repaired in an accelerated fashion, or apoptosis may occur in a more timely fashion.
The Environment can Put the Circadian Rhythm Out of Sync
Agents that interfere with one or more genes that regulate a cell's circadian rhythm can put a cell out of sync with the environment and with other cells. For example, in the hours following UV exposure, perhaps as many as 20 hours, the levels of clock, bmal1 and per1 gene expression in human keratinocytes are significantly depressed (see FIG. 1). By “significantly depressed” we mean below the minimum expression that these genes experience in their normal circadian cycle, as described above. Furthermore, following UV exposure, the usual pattern of gene expression, which may be described as roughly sinusoidal, is lost.
In FIG. 1, a horizontal line marks the usual average level of clock gene expression and, starting at about 44 hours following UV exposure, shows typical circadian variation about that line. In contrast, immediately following UVB exposure, levels of gene expression fell to well below normal, and did not return to normal levels for about 20 hours. During that 20 hours, the normal pattern of gene expression was lost for all three genes. And even when levels of gene expression did return to near normal levels, the period from about 20 hours to about 44 hours was required for the normal sinusoidal pattern of expression to return. So UV exposure really had two effects. One effect is the dramatic decline in the level of expression of circadian genes and the other is the pattern, which is to say, the timing of their expression. From 0 to about 44 hours following UV exposure, the timing of DNA and protein synthesis, mitosis and cytokinesis, RNA synthesis and repair and programmed cell death (apoptosis), are all compromised.
Of course, UV exposure occurs during the daytime, especially during the critical 10 a.m. to 2 p.m. window. As already noted, the concentration of PER and CRY proteins in the nucleus peaks at the end of circadian daytime, at which time the CRY proteins act to inhibit transcription of the CLOCK/BMAL1 heterodimer. Any delay in reaching the critical concentration that turns off clock and bmal1 expression, will lengthen the circadian cycle. So, UV exposure tends to lengthen the circadian cycle. This throws the cell cycle out of sync with the environment. DNA replication and mitosis may not occur at night, which is optimal for cell replication. Also, the gating influence that circadian genes exert on the cell cycle may be compromised, so that DNA damage may not be detected or may not be repaired, and may be allowed to pass on to daughter cells.
Sirtuins are Reported to Delay the Onset of Mitosis
Sirtuin 1 (also known as SIRT1 or Silent information regulator two ortholog 1) is an enzyme that regulates metabolism and cell survival in response to stress. It is associated with cell longevity. The sirt1 gene, which encodes for the SIRT1 enzyme, is not a circadian gene. Chua et al. have suggested that SIRT1 promotes replicative senescence by arresting the cell cycle (Chua et al. (2005) Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress. Cell Metabolism 2, 67-76).
US 2009-0082278 (herein incorporated by reference, in its entirety) further describes this gene and topical skin compositions that may upregulate it. Paragraphs 8-13 read:                “The applicants have recently discovered the involvement of a new protein in the mechanisms of skin cells which has a significant role in the aging process and cell protection.”        “The applicants have demonstrated that the SIRT protein, and more precisely the SIRT1 protein, was expressed in skin cells and that its expression was related to different stresses that cutaneous cells encounter. They have demonstrated in particular that the induction of this protein expression, using different agents, allowed for the protection of cells and better helped them to fight against stress and intrinsic aging.”        “SIRT proteins are part of the Sirtuin family, and are NAD+ dependent nuclear proteins that play a significant role in histone deacetylation. SIR genes (Silent Information Regulators), which code for SIR proteins, were described for the first time in S. cerevisiae in 1979 (Rine J and A l., Genetics 1979). Later, it was demonstrated that an over-expression of the SIR2P protein, in C. elegans, allowed the lifespan of the organism to increase (Tissenbaum and A l., Nature 2001). This study has permitted to hypothesize that these proteins are related to longevity.”        “The SIRT1 protein is the best characterized human sirtuin and interacts with numerous transcription regulators. The human SIRT1 protein has been described as being involved in p53 regulation (Cheng H L and A 1. Proc Natl Acad Sci USA. 2003), and more recently as a modulator of cell senescence (Langley E and A l., EMBO J. 2002). Other human SIRT proteins have been discovered (SIRT2, SIRT3, SIRT4-7). The human SIRT2 protein has been studied very little; however some studies have demonstrated its role in the control of mitotic activity (Dryden S C and A l. Mol Cell Bio. 2003) as well as its involvement in the regulation of the p53 protein (Vaziri H and A l., Cell. 2001). To date, deacetylase sirtuins are considered a family of enzymes playing an important role in the regulation of cellular death and in its lifecycle (Porcu M. and Chiarugi A, Trends Pharmacol Sci., 2005).”        “The present invention [that is, US 2009-0082278] relates to a cosmetic or pharmaceutical composition comprising, in a cosmetically or pharmaceutically acceptable medium, at least one compound likely to activate the synthesis of SIRT proteins in skin cells. Preferentially, according to the invention, the compounds will activate a particular class of SIRT proteins, the SIRT1 proteins.”        “To date, no use of compounds that serve as inductors of the synthesis of the SIRT family of proteins, in skin cells, has ever been described.”        
The present specification makes the point that, to date, no use of compositions comprising sirt1 activators in concert with circadian gene activators, has ever been described, even in US2009-0082278. To the best of the applicant's knowledge, a topical composition that addresses depressed levels of CLOCK and PER1 proteins in human epidermal keratinocytes, comprising one or more non-circadian, mitosis-delay agents, is unknown.
Topical Compositions for DNA Repair
Topical products for application to skin to promote the cellular repair process are known. For example, such products may include DNA repair enzymes for improving the effectiveness of natural cellular DNA repair, humectant ingredients for maintaining keratinocyte hydration, moisturizing ingredients for improving skin barrier function, and so on. While these ingredients may improve the ability of keratinocytes to repair themselves, there is always room for improvement. In contrast to the prior art, the present invention provides means of restoring the levels of undamaged circadian proteins in skin cells and means of restoring the normal pattern of circadian gene expression, combined with a means of inhibiting damaged DNA from being passed on to daughter cells.